Modified live Flavobacterium columnare against columnaris disease in fish

ABSTRACT

Safe and effective live vaccines against  Flavobacterium columnare  of fish were created through the induction of rifampicin resistance in a native  Flavobacterium columnare  isolate; these including rifampicin-resistant mutants NRRL B-30303 and B-30304. Single immersion exposure of fish stimulated acquired immunity against virulent  F. columnare  infection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Flavobacterium columnare is an aquatic bacterium that is highlyinfectious in both warm and cold water species of fish. In the channelcatfish (Ictalurus punctatus), it is the causative agent of columnarisdisease. Flavobacterium columnare is a Gram-negative, rod shaped,pathogen that has been isolated from channel catfish in areas of thesoutheastern United States where this species is cultured. The diseasealso affects sports fish (i.e., walleye and largemouth bass) andaquarium fishes. Medicated feed (antibiotics) is currently used to tryand control this bacterial infection. However, these treatments arelimited in their effectiveness and most producers have discontinued useof medicated feeds. Prevention of columnaris disease by vaccination isan important goal and a top priority of catfish and other fish producersthroughout the world. Estimated savings to these industries would be inexcess of $100 million annually.

This invention relates to a novel vaccine against columnaris which doesin fact provide superior protection over commercial treatment involvingtreating the water with potassium permanganate (KmnO₄) or feedingmedicated feeds.

2. Description of the Prior Art

It has been estimated that columnaris is the second leading cause ofmortality in pond raised catfish in the southeastern United States.Based on the success of disease control by immunization with killedbacteria (i.e., bacterins) in salmonids, experimental bacterins havebeen developed and tested against F. columnare. However, no vaccine iscurrently available and vaccination is not practiced in the catfishindustry against F. columnare, presumably because the inactivation(i.e., formalin treatment) destroys the antigen (Bader et al.,Comparison of whole-cell antigens of pressure-and formalin-killedFlexibacter columnaris from channel catfish (Ictalurus punctatus),American Journal of Veterinary Research, 58, pp 985-988, 1997). Thepatent of Wolf-Watz et al. (U.S. Pat. No. 5,284,653) presents a wholelist of bacteria of which one is Flexibacter columnaris (now F.columnare) which has the potential to be genetically modified to producea vaccine. However, no data are presented on F. columnare vaccines, onlyon genetically modified mutant vaccines of Vibrio anguillarum. Bernadet(Immunization with bacterial antigens: Flavobacterium and Flexibacterinfections, Fish Vaccinology: Developments in BiologicalStandardization, Volume 90, pp 335-340, 1997; Karger Switzerland: Basel)reviews the limited knowledge available on F. columnare and vaccinationagainst this important disease. Work suggests that rainbow trout(Oncorhynchus mykiss) which survive infection with F. columnare areimmune to subsequent disease.

SUMMARY OF THE INVENTION

We have now discovered a means for the creation of novel live vaccinesthat are safe and effective for the control of F. columnare in catfish.The vaccines comprise one or more rifampicin(3-[4-methylpiperazinyl-iminomethyl]rifamycin SV) (Sigma ChemicalCompany, St. Louis, Mo.) resistant mutants of F. columnare, created bymultiple passaging of the native isolate on increasing concentrations ofrifampicin. These vaccines are effective in providing long lastingacquired immunity in channel catfish to F. columnare.

In accordance with this discovery, it is an object of the invention toprovide a novel, highly protective, live vaccine against F. columnare infish, such as eels (Anguilla sp.), salmonids (Oncorhynchus sp. and salmosp.), tilapia (Oreochromis sp.), hybrid-striped bass (Morone chrysops×M.saxatilis), walleye (Stitzostedion vitreum), channel catfish, cetrachids(such as largemouth bass (Micropterus salmoides)), bait minnows(Pimephales promelas), goldfish (Carassius auratus), carp (Cyprinuscarpio), and aquarium fish (tropical fish species such as black mollies(Poecilia sphenops)) and platies (Xiphophorus maculatus).

It is another object to provide an attenuated F. columnare vaccine thatis safe and provides long lasting acquired immunity in fish tocolumnaris disease, including channel catfish.

It is a further object of this invention to improve the viability andproductivity of catfish, and to reduce economic losses in the fishindustry caused by columnaris disease.

Other objects and advantages of the invention will become readilyapparent from the ensuing description.

DEPOSIT OF BIOLOGICAL MATERIAL

Two rifampicin-resistant attenuated F. columnare isolates, weredeposited on Jun. 20, 2000 under the provisions of the Budapest Treatyin the Agricultural Research Service Culture Collection in Peoria, Ill.,and have been assigned Deposit Numbers B-30303 and B-30304.

DETAILED DESCRIPTION OF THE INVENTION

“Vaccine” is defined herein in its broad sense to refer to any type ofbiological agent in an administrable form capable of stimulating aprotective immune response in an animal inoculated with the vaccine. Forpurposes of this invention, the vaccine may comprise one or more liveattenuated mutants of F. columnare having the characteristic ofrifampicin-resistance.

The starting material for use in preparing the vaccines of the inventionis any attenuated F. columnare bacterium such as those reported, supra.Serial passage of the isolate of F. columnare over increasingconcentrations of rifampicin produces strains with an attenuatedpathogenicity efficacious for the preparation of live vaccines. Theattenuation achieved by high-level serial passage in culture onincreasing concentrations of rifampicin virtually eliminates thepathogenicity of the bacterium toward fish. The native strain of F.columnare should be passaged a sufficient number of times such that inits new attenuated form it no longer possesses the ability of causingthe disease state known as columnaris in catfish. The methodology forattenuation by serial passage is well known and documented in the art asexemplified by Schurig et al. [Vet. Micro. 28, 171-188 (1991)], herebyincorporated by reference, who created vaccines based on modified liverifampicin-resistant Brucella species.

Vaccination, while being accomplishable by injection or through oralingestion, is most efficiently done by means of aqueous immersion. Thebacterial agent is prepared for administration by formulation in aneffective immunization dosage with an acceptable carrier or diluent,such as water. The expression “effective immunization dosage” is definedas being that amount which will induce immunity in a fish againstchallenge by a virulent strain of Flavobacterium columnare. Immunity isconsidered as having been induced in a population of fish when the levelof protection for the population is significantly higher than that of anunvaccinated control group. One measure of protection followingexperimental challenge is relative percent survival (RPS) as describedby Amend (1981; Dev. Biol. Stand., 49, 447-454), herein incorporated byreference. RPS is calculated according to the following formula:${RPS} = {1 - {\frac{\%\quad{vaccinate}\quad{mortality}}{\%\quad{control}\quad{mortality}} \times 100}}$

A positive vaccinal effect is indicated by a RPS equal to or greaterthan 60%. Typically, vaccination is carried out by exposing fish byimmersion in water containing about 1×10⁶ CFU/ml of attenuatedFlavobacterium columnare for 15 minutes at a density of about 50 fish/Land a temperature of about 25° C. CFU denotes colony forming units of F.columnare. These parameters may be varied as desired such that asufficient level of vaccination is acquired without induction ofstressful conditions or loss of fish. Useable concentrations ofFlavobacterium columnare are considered to range from about 5×10⁵ toabout 1×10⁸ CFU/ml of immersion medium. Useable vaccination times areseen to range from about 1 minute to about 60 minutes, preferably fromabout 2 minutes to about 15 minutes. Temperature of the inoculationmedia may range within the physiologically acceptable limits of the fishinvolved, for channel catfish preferably from about 18° C. to about 28°C., most preferably from about 22° C. to about 26° C. Concentrations offish treated in the inoculation medium typically range from about 50 toabout 100 fish/L, but, in the alternative, may be determined on a weightbasis and range from about 0.5 to about 2.5 kg/L. The vaccine can beeffectively administered any time after the fish attainsimmunocompetence, which for channel catfish is at about two to fourteendays post-hatch. Other species of fish susceptible to F. columnare canbe immunized after 21-30 days post-hatch or when they becomeimmunocompetent to modified live vaccine administered by immersion.

Appropriate adjuvants as known in the art may also be included in thevaccine formulation.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims.

EXAMPLE 1

The procedure used to produce the F. columnare vaccine mutants wasmodified from that described in Shurig et al. (1991; Vet Micro, 28,171-188), hereby incorporated by reference, by using a lower initialconcentration of rifampicin and ending at 200 μg/ml after 243 passagesinstead of 51 passages and by omitting the penicillin step.

Process of Developing Resistant Mutants of Flavobacterium columnare

Modified Cytophaga agar plates for the cultivation of Flavobacteriumcolumnare were made according to the procedure of Klesius et al. (Effectof feed deprivation on innate resistance and antibody response toFlavobacterium columnare in channel catfish, Ictalurus punctatus.Bulletin European Association of Fish Pathologists, 19(4), 156-158,1999). 1.0 g tryptone, 0.5 g yeast extract, 0.2 g beef extract, 0.2 gsodium acetate and 9.0 g of agar were added to one liter of distilledwater. The medium was heated until dissolution. The medium was thenautoclaved at 121-124° C. for 15 minutes, poured into sterile petridishes (15 ml per dish) and allowed to solidify before refrigeration.

Native isolates of Flavobacterium columnare were obtained from sickcatfish or previously obtained lyophilized stocks. Isolates of F.columnare were then identified by standard biochemical tests as setforth in Bergey's Manual of Determinative Bacteriology prior to use inrifampicin resistant F. columnare. After identification, the process offorming rifampicin resistant isolates of F. columnare was begun.Rifampicin supplemented modified Cytophaga agar plates were prepared asfollows: Modified Cytophaga agar was made as described above andsterilized at 121-124° C. for 15 minutes. After sterilization, thecorrect amount of rifampicin was added to the media prior to itssolidification and 15 ml of the resulting mixture was poured intoseparate petri dishes and allowed to solidify prior to refrigeratedstorage. Initial cultures of the native isolates of F. columnare weregrown on modified Cytophaga agar plates which were incubated at 20-25°C. for 24-48 hours or until 1-2 mm yellow, rhizoid colonies wereobserved. A single F. columnare colony was then picked with a sterileinoculating loop and streaked onto a rifampicin supplemented modifiedCytophaga agar plate containing the correct concentration of theantibiotic. For the initial passage, rifampicin was present in themodified Cytophaga agar at a concentration of 5 μg/ml. Therifampicin-supplemented modified Cytophaga agar which was streaked withthe aforementioned native isolate of F. columnare was then incubated for24-48 hours at 20-25° C. and observed for bacterial growth. Singlecolonies of F. columnare which grew on the rifampicin-supplementedmedium were then picked and placed onto the next concentration ofrifampicin (10 μg/ml) modified Cytophaga agar plates. If growthoccurred, a single colony was harvested and placed on an agar mediumcontaining the next higher concentration of rifampicin (20 μg/ml). Ifthe harvested colony failed to grow, it was repeatedly passed on amedium containing the last concentration of rifampicin at whichgrowth'successfully occurred, before being placed on the next higherconcentration of rifampicin-containing medium. This process was repeateduntil a colony capable of growing on a medium containing a rifampicinconcentration of 200 μg/ml was created.

Flavobacterium columnare isolate ARS-1 was passaged on increasingconcentrations of rifampicin (Sigma Chemical Company, St. Louis, Mo.)supplemented modified Cytophaga agar to a final concentration of 200μg/ml rifampicin for 243 passages. The resultant mutants (i.e., twocolonies from the original passage that grew and were passaged),designated B-30303 and B-30304, are differentiated from the parentmicroorganism because they can survive and reproduce on a mediumcontaining 200 μg/ml rifampicin without negative effect. Biochemicalcharacteristics of the F. columnare B-30303 and B-30304 are identical toF. columnare as described in Bergey's Manual of DeterminativeBacteriology (Holt et al., 1994), herein incorporated by reference.

EXAMPLE 2 Safety and Back-passage

The safety and back-passage study revealed both vaccine candidaterifampicin mutants were safe for use in vaccination of 10 day post hatchchannel catfish at concentrations of 1.4×10⁸ or 6.7×10⁷ CFU/ml for a 30minute immersion exposure. One hundred and twenty five fish were usedfor each group which included the initial exposure and then eachsubsequent exposure for a total of 5 passages (i.e., fish to fishtransfer). One hundred channel catfish not exposed to F. columnaremutants were kept as negative controls. The numbers of fish which diedafter exposure to the vaccine or vaccine diluent (i.e., controls) arepresented in Table 1. Forty eight hours following exposure, 40 fish wereremoved, homogenized and cultured for the presence of F. columnarevaccine strains at each passage and in the control group. The vaccineisolates were isolated at the first passage but not at subsequentpassages indicating that the vaccine strains were capable of invadingthe fish, but did not revert to virulence following back-passage andthus, were attenuated in 10 day post hatch channel catfish. The fishused in this experiment were held in the laboratory without signs ofcolumnaris disease or adverse behavior for at least 21 days followingtreatment.

TABLE 1 SAFETY OF VACCINE DOSE OF F. COLUMNARE MUTANTS IN 10 DAY POSTHATCH CHANNEL CATFISH VACCINATES F. columnare Mutant No. Dead/ Isolatedfrom Percent Mutant Passage No. Total¹ homogenized fish MortalityB-30303 1 14²/125   Yes 11.2 2 0/125 No 0.0 3 0/125 No 0.0 4 0/125 No0.0 5 0/125 No 0.0 B-30304 1 17³/125   Yes 13.6 2 1⁴/125  No 8.0 312⁵/125   No 9.6 4 2⁶/125  No 1.6 5 0/125 No 0.0 Controls None 13⁷/100  No 13.0 ¹Note, total number of fish to start the experiment was used forcalculations. ²1 of 14 positive for rifampicin resistant F. columnare.³1 of 17 positive for rifampicin resistant F. columnare. ⁴ F. columnarenot isolated from dead fish. ⁵ F. columnare not isolated from dead fish.⁶ F. columnare not isolated from dead fish. ⁷ F. columnare not isolatedfrom dead fish.

EXAMPLE 3 Efficacy

Channel catfish (125, USDA 103 strain) were vaccinated by adding 40 mlof F. columnare mutant B-30303 to 7.5 L of water for a 30 minuteexposure. One-hundred twenty five USDA 103 channel catfish were exposedby adding 40 ml of vaccine diluent to 7.5 L of water for a 30 minuteexposure to serve as control fish. (i.e., non-vaccinated). Vaccinatedand control fish were held for 32 days following vaccination before theywere challenged with virulent F. columnare. No fish died after thevaccination. Results of experimental challenge are presented as relativepercent survival (RPS) as described by Amend (1981; Dev. Biol. Stand.,49, 447-454), herein incorporated by reference. RPS is calculatedaccording to the following formula:${RPS} = {1 - {\frac{\%\quad{vaccinate}\quad{mortality}}{\%\quad{control}\quad{mortality}} \times 100}}$

A positive effect by vaccination is a RPS greater than 50%. At fourteendays post vaccination (DPV), the relative percent survival (RPS) was69.2%. In this study, mortality in an equivalent group of 125 untreatedcontrols was 20.8% versus 6.4% mortality in the 125 vaccinated fish.

TABLE 2 PROTECTION AGAINST COLUMNARIS DISEASE AFTER IMMERSIONVACCINATION¹ OF CHANNEL CATFISH WITH FLAVOBACTERIUM COLUMNARE B-30303VACCINE No. Dead/ Percent Relative Percent Treatment No. Total MortalitySurvival (RPS)² Vaccinated with  8/125 6.4 69.2 B-30303 Control 26/12520.8 — (non-vaccinated) ¹Immersion vaccination for 30 minutes with 5 ×10⁶ CFU/ml F. columnare B-30303 for 30 minute immersion exposure.²Relative percent survival as determined by Amend (1981).

EXAMPLE 4

Fish were vaccinated (48 day post hatch channel catfish) with themodified live F. columnare B-30303 for two minutes at the describedvaccine dose and then diluted with an equal volume of water and held foran additional 13 minutes (total vaccine time 15 minutes). Control andcontrol non-challenged groups were immersed in vaccine diluent for thesame duration of exposure (15 minutes). The fish were challenged 57 daysfollowing vaccination by co-habitation following 14 days without feed(Klesius et al., 1999). Three channel catfish showing signs ofcolumnaris disease were added to each of the groups A, B and C andallowed to remain with fish for 24 hours. A sub-sample of five dead fishof those used for cohabitation were culture positive for F. columnare.The dead fish were removed at 24 hours the following day and feedingresumed. Table 3 shows the relative percent survival was calculatedaccording to Amend (1981).

TABLE 3 PROTECTION AGAINST COLUMNARIS DISEASE AFTER IMMERSIONVACCINATION OF CHANNEL CATFISH WITH FLAVOBACTERIUM COLUMNARE B-30303VACCINE Mean % Relative No. Dead/ Mortality Percent Group Treatment No.Total (SEM¹) Survival² A Vaccinated 2/150 1.33 96.4 1 × 10⁶ CFU/ml(0.67)^(a) B Vaccinated 7/150 4.67 97.3 5 × 10⁶ CFU/ml (0.67)^(a) CControl 55/150  36.67 — (17.7)^(b) D Control 3/150 2.00 — Non-challenged(1.15)^(a) ¹Means with different superscripts are significantlydifferent at p < 0.05 using Duncan's Multiple range test for differences(SAS Inc., 1997). ²Relative percent survival calculated according toAmend (1981).

EXAMPLE 5

Flavobacterium columnare isolates were obtained from channel catfishwith signs of columnaris disease. The F. columnare were cultured onCytophaga agar and determined to be pure cultures. Isolates obtainedwere frozen in 2 ml aliquots at −80° C. Once thawed to 25° C., fiftymicroliters were then plated onto each of the media types (i.e.,Cytophaga agar and Cytophaga agar supplemented with 5 μg rifampicin/ml)and incubated at 25±3° C. for 24 hours. The development of yellowrhizoid colonies on agar was considered positive for growth. The ten F.columnare isolates tested grew on Cytophaga agar only with typical F.columnare colonies developing. Growth was not observed with any of theisolates tested on Cytophaga agar supplemented with 5 μg rifampicin/ml.

Culture was also attempted after the isolates recovered from coldstorage had grown on the non-rifampicin supplemented plates for 48hours. Single colonies of the recovered bacteria that were growing onCytophaga agar were picked and streaked onto Cytophaga agar supplementedwith 5 μg rifampicin/ml. No growth occurred on Cytophaga agarsupplemented with 5 μg rifampicin/ml demonstrating that these isolateswere not viable candidates for development of modified live F. columnarevaccines because rifampicin resistance could not be induced in thesenative F. columnare isolates (see Table 4). These results support theunexpected nature of the success achieved in Example 1.

TABLE 4 Failure of ten Flavobacterium columnare isolates tested to growon rifampicin supplemented Cytophaga agar (5 μg ritampicin/ml) Growth onGrowth on Cytophaga Isolate Cytophaga rifampicin supplemented agarIdentification¹ Agar (5 μg rifampicin/ml) AUFAA-1² Yes No Growth AUFAA-2Yes No Growth AUFAA-3 Yes No Growth AUFAA-4 Yes No Growth AUFAA-5 Yes NoGrowth ALG-530³ Yes No Growth ALG-515 Yes No Growth ALG-513 Yes NoGrowth ALG-527 Yes No Growth ALG-521 Yes No Growth ¹ Flavobacteriumcolumnare isolates all from channel catfish showing signs of columnarisdisease. ²AUFAA = Auburn University Fisheries and Allied Aquaculture -Isolate Number. ³ALG = Alabama Fish Farming Center, Greensboro, AL -Case Number.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modification and variations may be madetherein without departing from the spirit and scope of the invention.

1. An attenuated strain of Flavobacterium columnare resistant torifampicin and effective for eliciting an immune response in fish whichis protective against infection by virulent strains of Flavobacteriumcolumnare wherein said strain of Flavobacterium columnare is selectedfrom the group consisting of NRRL B-30303 and B-30304.
 2. The attenuatedstrain as described in claim 1 wherein said strain of Flavobacteriumcolumnare is NRRL B-30303.
 3. The attenuated strain as described inclaim 1 wherein said strain of Flavobacterium columnare is NRRL B-30304.4. A vaccine for protecting fish against infection by virulent strainsof Flavobacterium columnare comprising: (1) in a dosage effective toprotect fish against infection by virulent strains of Flavobacteriumcolumnare the attenuated strain of Flavobacterium columnare of claim 1;and (2) a carrier.
 5. The vaccine as described in claim 4 wherein saidstrain of Flavobacterium columnare is NRRL B-30303.
 6. The vaccine asdescribed in claim 4 wherein said strain of Flavobacterium columnare isNRRL B-30304.
 7. The vaccine as described in claim 4 wherein saidcarrier is water.